Microglia undergo metabolic adaptation during heme detoxification

Intracerebral hemorrhage (ICH) is a devastating form of stroke that results in significant mortality, and survivors typically suffer from lasting neurological deficits. Despite the severity of ICH and its complications, there are currently no therapies specifically targeted to reduce oxidation-induced damage after ICH. After ICH, damaged erythrocytes release heme into the surrounding environment, where it promotes oxidative damage and inflammation. Microglia, the tissue-resident macrophages of the central nervous system, are responsible for clearing the heme released after ICH. It has been shown that knockdown of heme clearance enzymes, including Heme Oxygenase 1 (HMOX1), in microglia worsens outcome in models of ICH, but the role of metabolic adaptation in microglial heme clearance remains unexplored. Since improving the rate of heme clearance through metabolic manipulation might prove a potential avenue for ICH therapy, we sought to fully understand the interplay between metabolism and heme clearance in heme-exposed microglia. Here we show that heme rapidly alters the metabolic profile of BV2 murine microglia, increasing glycolytic rate at low doses (1-10μM) and decreasing oxygen consumption at higher doses (50μM) using extracellular flux analysis. We also show that subsequent treatment with Ferrostatin-1, an antioxidant shown to prevent iron-dependent cell death (“ferroptosis”), rescues the heme-induced decrease in oxygen consumption. Heme also upregulates expression of both Hmox1 and the Pentose Phosphate Pathway (PPP) genes, Glucose-6-phosphate dehydrogenase (G6pd) and 6-Phosphogluconate dehydrogenase (Pgd), in a dose-dependent manner by Reverse Transcription-Quantitative Polymerase Chain Reaction (RTqPCR). We show that intracellular accumulation of heme in microglia is also dose-dependent and occurs rapidly after heme exposure. Additionally, pharmacologic inhibition of fatty acid and glutamine flux into the tricarboxylic acid (TCA) cycle decreased the rate of heme loading and degradation. Together, these data suggest that metabolic manipulation of microglia is a potential avenue to improve heme clearance and prevent oxidative damage after ICH.